In this study, the oestrogen agonist/antagonist action of 4-hydroxytamoxifen (OHT; 1!10 K6 M) and 17b-oestradiol (E 2 ; 1!10 K8 M) were assessed on the oestrogen receptor (ER)-positive epithelial cell line (Ishikawa) with respect to cell proliferation, and to gene and protein expression. qRT-PCR and western blotting confirmed that Ishikawa cells expressed both ER isoforms and that there was no change in transcript levels in response to either ligand. Gene expression profiles, using oligonucleotide arrays representing w19 000 human genes, showed that the expression of 716 and 534 genes were changed differentially by treatment with either OHT or E 2 respectively, at the 24-h time point, with modulation of 46 genes common to both ligands, whereas 335 (OHT) and 240 (E 2 ) genes showed expression changes unique to ligand, with 13 common alterations at 48 h. Both OHT and E 2 had demonstrable oestrogen agonist actions on Ishikawa cells, exemplified by increased proliferation and expression of known oestrogen-responsive genes, such as creatine kinase B and by the induction of alkaline phosphatase activity. Additionally, the data indicate that the two oestrogen agonists generated not only common gene expression changes but also unique ligand-specific profiles, raising the intriguing possibility that tamoxifen has E 2 -independent effects on the uterine epithelium.

Tamoxifen acts as an oestrogen antagonist in the breast reducing cell proliferation, but in the uterus as an oestrogen agonist resulting in increased cell proliferation. Tamoxifen exerts its tissue-specific effects through the oestrogen receptors (ER or ER ). The levels and functions of the two ERs affect the response of the target tissue to oestrogen and tamoxifen. We examined the control of ER stability in breast and uterine cell lines using western blotting and RT-PCR. In MCF-7 breast-derived cells, ER and ER proteins were rapidly degraded via the proteasome pathway in response to oestradiol; conversely tamoxifen stabilised both receptors. In Ishikawa uterine-derived cells, oestradiol and tamoxifen stabilised ER but led to degradation of ER by the proteasome pathway. Further investigations showed that oestradiol induced activation of the non-genomic ER /Akt signalling pathway in MCF-7 cells. We have demonstrated that the alternative Erk signalling pathway is activated in Ishikawa cells following oestradiol treatment in the absence of an active proteasome pathway and therefore increased levels of ER . In conclusion, our data have demonstrated tamoxifen or oestradiol control of ER subtype stability and that non-genomic activation of transcription pathways is cell specific.